Synthesis and molecular docking simulations of novel azepines based on quinazolinone moiety as prospective antimicrobial and antitumor hedgehog signaling inhibitors

A series of novel azepine derivatives based on quinazolinone moiety was synthesized through the reaction of quinazolinone chalcones (2a–d) either with 2-amino aniline in acidic medium to give diazepines (3a–d) or with 2-aminophenol to offer oxazepine (4a–d). The structure of the synthesized compounds was confirmed via melting points, elemental analyses, and different spectroscopic techniques. Moreover, these newly compounds mode of action was investigated in-silico using molecular docking against the outer membrane protein A (OMPA), exo-1,3-beta-glucanase for their antimicrobial activity, and against Smoothened (SMO), transcription factor glioma-associated homology (SUFU/GLI-1), the main proteins of Hedgehog signaling pathway to inspect their anticancer potential. Our results showed that, diazepine (3a) and oxazepine (4a) offered the highest binding energy against the target OMPA/ exo-1,3-beta-glucanase proteins and exhibited the potent antimicrobial activities against E. coli, P. aeruginosa, S. aureus, B. subtilis, C. Albicans and A. flavus. As well, diazepine (3a) and oxazepine (4a) achieved the best results among the other compounds, in their binding energy against the target SMO, SUFU/GLI-1 proteins. The in-vitro cytotoxic study was done for them on panel of cancer cell lines HCT-116, HepG2, and MCF-7 and normal cell line WI-38. Conclusively, it was revealed that molecular docking in-silico simulations and the in-vitro experiments were agreed. As a result, our findings elucidated that diazepine (3a) and oxazepine (4a), have the potential to be used as antimicrobial agents and as possible cancer treatment medications.

The World Health Organization (WHO) estimates that antimicrobial resistance and cancer incidence remain the major concern diseases despite advances in preclinical and clinical research, due to a variety of heterogeneous risk factors including ethnicity, environmental exposure, gender, socioeconomic factors, genetic predisposition, location, and dietary habits 1 .
Antimicrobial resistance is influenced by outer membrane protein A (OMPA) and exo1,3 beta glucanases 2 .OMPA has a variety of roles in the pathophysiology of bacteria, including resistance, induction of host cell death, and adhesion to host cells.Clinically, overexpression of the OMPA gene is linked to the onset of pneumonia and bacteremia, as well as patient death 3 .Furthermore, β-1,3-glucanases is the primary skeletal polysaccharides of fungal cell walls that catalyzes the hydrolytic cleavage of the β-1,3-D-glycosidic linkages in β-1,3-glucans and it is the key enzyme in the lysis of phytopathogenic fungal cell walls during the pathogenicity, which appears to be the primary role for treatment 4. General procedure for the synthesis of chalcones (2a-d) Equimolar amounts (10 mmol) of 3-(4-acetylphenyl)-2-phenylquinazolin-4(3H)-one (1) and different aromatic aldehydes were dissolved in ethanol (15 mL).Sodium hydroxide (0.08 g, 2 mmol) was added and stirred for 24 h.The reaction mixture was poured in to crushed ice, filtered, washed, and dried.

In-silico ADMET prediction
The online tool SwissADME (http:// www.swiss adme.ch/) from the Swiss Institute of Bioinformatics was utilized to investigate the pharmacokinetics and drug-likeness prediction of the newly synthesized compounds.The compound's 2D structural model was converted into SMILES using SwissADME's SMILES generator.The SMILES data was then examined to identify the compound's ADMET properties, including pharmacokinetics and drug-likeness 24,25 .
Vol Antimicrobial activity testing of the novel azepine derivatives using agar well diffusion method.By using the agar well diffusion technique, the diameter of the inhibitory zone was determined to evaluate how susceptible the tested bacterial and fungal strains were to the synthetic novel azepine derivatives.The synthesized azepine derivatives were prepared in DMSO at a concentration of 10 mg/mL antibiotic Ciprofloxacin, antifungal Clotrimazole and DMSO were used as positive and negative controls, respectively, to compare the effectiveness of bacterial and fungal strains.Before being adjusted to 106 CFU/mL at 630 nm, the bacterial and fungal strains underwent an overnight sub-culture in a nutrient broth medium.A 100 μL aliquot of each broth culture was evenly seeded throughout the nutrient agar medium using a sterile disposable plastic rod.On the surface of the nutritional agar medium, 9 mm wells were successfully made using a sterile cork porer, and 50 μL of each compound was then added 26 .The % activity index for the complex was calculated by the formula as follow: Minimal inhibitory concentration (MIC).The azepine derivatives were next evaluated in DMSO at different dosages (0.5, 3.75, 7.5, 10 mg/mL) to observe their antimicrobial properties.The examined bacterial or fungal strains were placed in a loop that was submerged in 10 mL of nutrient broth and grown at 30 °C overnight.Test tubes were prepared and sterilized with 9.5 mL of 10 × diluted nutritional broth.The tubes were inoculated with 0.5 mL of the suitable microbe that had been cultured overnight.The chosen bioactive azepine derivatives were added to the tubes containing the nutrient broth.A shaking incubator was used to stir the cultures of the test organisms and the synthesized azepines at various concentrations at 30 °C.Following 24 h, the number of living cells was determined as colony-forming units/milliliter (CFU/mL) in accordance with Nakashima et al. 's instructions 27 .
Cells treatment and viability assay.Different concentrations (0-200 μM) of both the newly azepine derivatives and the reference inhibitory Hedgehog GANT-61 drug were applied to the cells.MTT (5 mg/mL in PBS) was applied to each well after 48 h of incubation, and the cells were then cultivated for an additional 4 h at 37 °C in a cell culture incubator.100 μL of DMSO was added to the wells after supernatant aspiration and shaken for 15 min.Using a microplate reader (Bio-Rad, CA, USA), the absorbance was found at 630 nm 17 .

Statistical analysis
The experimental data were expressed as the mean ± SE, and the IC 50 values were calculated Nonlinear regression curve fit (dose-response inhibition) using GraphPad Prism software 6 (San Diego, CA) (https:// www.graph pad.com/ scientific-software/ prism/).

Chemistry of the synthesized compounds
A series of chalcones (2a-d) were synthesized through the one pot reaction between 3-(4-acetylphenyl)-2-phenylquinazolin-4(3H)-one (1) with different aromatic aldehydes as illustrated in Fig. 2. The structure of these compounds was confirmed via elemental analysis and different spectroscopic data.According to Fig. 3, the reaction of chalcones (2a-d) with 2-amino aniline offers the diazepine derivatives (3a-d).Their chemical structures were demonstrated via both elemental analysis and different spectral data.The FT-IR spectra showed an absorption band at 1537-1611 cm −1 which characterized to C=N of diazepine ring and at 3300-3630 cm −1 for NH group which appeared as a singlet signal at δ 10.65-11.72 ppm in 1 H-NMR spectra.Revealed a new signal resonated at δ 3.35-4.42ppm due to CH proton of diazepine ring and a doublet signal at δ 1.55-2.18ppm attributed to CH 2 group. 13 Further modification of compound 1 with o-aminophenol offered oxazepine derivatives (4a-d) as illustrated in Fig. 4. Their FT-IR spectra indicated the appearance of absorption band at 1540-1598 cm −1 for C=N of oxazepine ring.The 1 H-NMR spectra offered a signal at δ 4.10-4.50ppm resonated to CH proton of oxazepine ring and doublet signal at 1.65-1.91ppm for CH 2 group. 13C-NMR spectra offered signal at δ 170.50-171.12,δ 61.95-84.10 and δ 31.12-36.20 for C=N, CH and CH 2 respectively in the oxazepine ring.

% Activity Index =
Zone of inhibition by test compound (diametre) Zone of inhibition by standard(diametre) × 100

In vitro biological assessments
Antimicrobial studies Resistant strains have evolved as a significant threat to population health and the global economy because of reckless antibiotic usage and inadequate infection management.As a result, it is critical to conduct extensive research and develop a new class of antimicrobial compounds to halt the spread of antimicrobial resistance (AMR) 33 .According to the in-silico results, diazepine (3a) and oxazepine (4a) were evaluated for in-vitro antibacterial activity against Gram-positive bacteria S. aureus (MTCC-96) and B. subtilis (MTCC-441), Gramnegative bacteria E. coli (MTCC-614) and P. aeruginosa (MTCC-1035) and fungal C. albicans (MTCC-3017) and A. flavus (MTCC-227) using a standard agar well diffusion method and inhibitory zone diameters (mm) are summarized in Table 3; Fig. 10.Our results evaluated that diazepine (3a) and oxazepine (4a) elucidated strongest antimicrobial effect, inhibiting the growth of all the investigated microorganisms.These compounds Moreover, the smallest amount of the target diazepine (3a) and oxazepine (4a) required to inhibit microbial growth is referred to as the minimum inhibitory concentration (MIC).Drug formulations can benefit greatly from this approach.The ratio of surviving cell numbers was then assessed to estimate the level of antimicrobial activities of the diazepine (3a) and oxazepine (4a) (Table 4).diazepine (3a) and oxazepine (4a) exhibited reasonable biocidal activity at very low concentrations against the tested microorganisms compared with the reference drugs.

In-vitro Antineoplastic cytotoxic studies
The antitumoral investigations using MTT assay were carried out to confirm the capability of diazepine (3a) and oxazepine (4a) in suppressing the aberrant of hedgehog (HH-GLI) signaling pathway, these azepine derivatives were selected among the others due to their highest binding energies in the in-silico studies.The MTT assay is a typical colorimetric method for evaluating cell growth.It is used to assess the cytotoxicity of other hazardous compounds and potential therapeutic medications.shortly, the enzyme mitochondrial dehydrogenases convert the yellow MTT into the purple formazan in living cells.By incorporating a suitable solvent, this formazan product is dissolved into a vibrant solution 34 .The precise concentration of the colored solution can be determined by measuring it at a particular wavelength.By plotting a dosage response curve and comparing the quantity of purple formazan that treated cells and untreated control cells generate, it is possible to evaluate how effectively the newly azepine derivatives destroy cancer cells.Our results elucidated that the novel diazepine (3a) and oxazepine (4a) observed significant (p < 0.001) antitumor effect against panel of cancer cells (HCT-116, HepG-2 and MCF-7) compared with the reference HH-GLI inhibitory drug GANT-61.Moreover, the most crucial step in evaluating the anti-cancer effects of newly azepine derivatives is to determine their cytotoxicity on normal cell lines.Here, we used the human normal lung fibroblast (WI-38).Our findings showed that none of our newly synthesized azepine derivatives have any cytotoxicity on normal cells (p < 0.0001), in contrast to GANT-61, which showed moderate toxicity towards normal cells (p < 0.001) (Fig. 11; Table 5).The newly synthesized diazepine (3a) and oxazepine (4a) could therefore be employed as promising anticancer drugs by inhibiting HH-GLI signaling pathway.Therefore, the MTT results supported the outcomes of the molecular docking simulations.
The presence of hetero atoms like oxygen and nitrogen in the chemical structure of the azepines enhanced the value of binding energy through the interaction between the azepines and the target protein via hydrogen bonding.
Compounds 3a and 4a were considered the most active compounds due to the presence of phenyl group and olefinic protons which enhanced the hydrogen bonding with the target protein and hence increased the value of the binding energy 18 .

Conclusion
In conclusion, quinazolinone chalcones (2a-d) were synthesized and used for the synthesis of novel diazepines (3a-d) and oxazepines (4a-d).The synthesized compounds were characterized by elemental analysis and different spectroscopic data.The results of novel diazepine (3a) and oxazepine (4a) antimicrobial effectiveness against six Gram-negative, positive multidrug-resistant bacterial isolates and unicellular pathogenic fungal strains showed a broad spectrum of their biocidal activity, which was consistent with their in-silico binding energies against OMPA and exo-1,3-beta-glucanase target proteins.Moreover, a substantial anticancer effect of diazepine (3a) and oxazepine (4a) were also observed against panel of cancer cell lines via suppressing hedgehog (HH-GLI) signaling pathway, which was supported by their molecular docking investigations against the SMO and SUFU/ GLI-1 target (HH/GLI) proteins.Overall, it is recommended to use these novel diazepine (3a) and oxazepine (4a) as potential antimicrobial and anticancer agents in medical applications.Antibiotic ciprofloxacin reference drug 0.5 ± 0.12 1 ± 0.05 0.5 ± 0.062 2 ± 0.   Asperlicine C (marketed anti cancer drug) [36] Raltitrexed (marketed anti cancer drug) [37] Albaconazole (marketed anti microbial drug) [37] Figure 12.Structure activity relation of the novel synthesized azepine derivatives.

Figure 1 .
Figure 1.The rational design of the newly azepine derivatives based on quinazolinone moiety as antimicrobial and anticancer agents.

Biological evaluations (in vitro)
Antimicrobial assessmentsThe clinical strains utilized in this experiment were graciously donated by the clinical laboratory at Tanta University Hospital in Tanta, Egypt.Four multidrug-resistant Gram −ve and Gram + ve bacterial strains E. coli, P. aeuroginosa, S. aureus, B. subtilis and two pathogenic unicellular fungi C. Albicans and A. flavus were used.The azepine derivatives which show the best inhibitory binding energies in the in-silico studies were proceeded for further antimicrobial investigations. .:(0123456789)

Table 1 .
Calculated docking scores (kcal/mol) of compounds 3a and 4a and reference drugs with the target proteins.